Nerve growth factor production accelerators and compositions for preventing or treating neuronal degeneration

ABSTRACT

Nerve growth factor production accelerating agents containing oxazopyrroloquinolines, pyrroquinolinequinones and/or their esters as active ingredient are provided. As the oxazopyrroloquinolines and their esters exhibit such production accelerating activity, they are suitably utilized for preventing and treating functional disorders of central nervous system, particularly, Alzheimer&#39;s dementia, cerebral ischemia and spinal trauma, as well as for functional disorders of Peripheral nervous system, particularly, peripheral nervous system trauma and diabetic neurosis. As the pyrroquinolineguinones and their esters exhibit strong nerve growth factor production accelerating activity, they are suitably utilized for preventing and treating functional disorders of peripheral nervous system, particularly, peripheral nervous system trauma, diabetic neuropathy, etc.

This application is a continuation of application Ser. No. 08/009,806, filed Jan. 27, 1993, now abandoned.

This invention relates to pharmaceuticals, particularly, those for treating or preventing retrograde neural diseases, such as dementia senilis and Alzheimer's disease, and further to production accelerators for nerve growth factor (hereinafter referred to as NGF) which works for the recovery of neural function in central and peripheral nervous system diseases. Furthermore, it relates to compositions for preventing or treating neuronal degeneration.

NGF is a nutrition and growth factor necessary for the growth and maintenance of neuronal tissues, which is considered to be essential for maturation and differentiation of sensory and sympathetic nerves in the peripheral nervous system, and magnocellular cholinergic neurons in the central nervous system, as well as for life maintenance. It has, thus, been thought that the increase in the NGF level serves for the treatments of central functional disorders, such as Alzheimer's disease, vascular dementia and spinal trauma and peripheral functional disorders, such as peripheral nervous trauma and diabetic neuronal disorders.

However, NGF is a protein with the molecular weight of 13,000 as monomer and 26,000 as dimer, so that it can not pass through the blood-brain barrier. Accordingly, it has been thought preferable that an agent accelerating the product ion of NGF in the living body, rather than NGF itself, is administered to promote biosynthesis of NGF, thereby to improve disorders of the central and peripheral nervous systems. Thus, investigations to seek NGF production accelerators have been attempted.

Cathecholamines, such as epinephrine, norepinephrine and dopamine, have been found as agents having NGF production accelerating activity. However, because these compounds are a kind of hormones, their administration to accelerate the NGF synthesis is accompanied by some side effects due to quantitatively ill-balanced hormone in the living body. Therefore, satisfactory drugs have not yet been discovered from the practical point of view.

The present inventors have extensively studied on NGF production accelerating agents on account of the reasons as mentioned above, and accomplished the present invention, based upon the findings that oxazopyrroloquinolines, pyrroloquinolinequinones, and their esters exhibit NGF production accelerating activity. Accordingly, the present invention provides an NGF production accelerating agent containing an oxazopyrroloquinoline or pyrroloquinolinequinone and/or their esters as an active ingredient.

The term, oxazopyrroloquinolines (hereinafter referred to as OPQs), used herein means 2,8,10-tricarboxy-1H-oxazo[5,4-h]-pyrrolo[2,3-f]quinolines (OPQ) and 5-substituted compounds thereof. The OPQs and their esters are represented by the following formula: ##STR1## wherein R represents a hydrogen atom or an alkyl group having 1-4 carbon atoms, which may be substituted with a hydroxyl, carboxyl, mercapto, carbamoyl, hydroxyphenyl, guanidyl, imidazolyl, or methylmercapto group, and R¹, R² and R³ represent a hydrogen atom or an alkyl, alkenyl or benzyl group which may be same or different.

OPQs employed in the present invention can readily be prepared by a process wherein a pyrroloquinolinequinone compound or its salt (hereinafter referred to as PQQ, which will be explained more fully below) is allowed to react with an α-amino acid, methylamine or the like in the presence of oxygen. The reaction is usually undertaken in a aqueous medium like a microbial culture. The pH of the reaction mixture is usually in the range from 2 to 10, and the reaction temperature is practically in the range from 20° to 100° C. The reaction time is preferably within 24 hours.

OPQs in the present invention include OPO (R═H) obtained from a PQQ and any of glycine, threonine, tryptophan, proline, tyrosine, serine, and monomethylamine [Japanese Patent Publication (Laid-Open) No. 294281/1991]; hydroxymethyl-OPQ obtained from a PQQ and serine [Japanese Patent Publication (Laid-Open) No. 123782/1991]: 1-methylethyl-OPQ obtained from a PQQ and valine [Japanese Patent Publication (Laid-Open) No. 170484/1991]; 1-methylpropyl-OPQ obtained from a PQQ and isoleucine [Japanese Patent Publication (Laid-Open) No. 170485/1991]; 2-methylpropyl-OPQ obtained from a PQQ and leucine [Japanese Patent Publication (Laid-Open) No. 170486/1991]; methyl-OPQ obtained from a PQQ and alanine [Japanese Patent Publication (Laid-Open) No. 188081/1991]; 2-carboxyethyl-OPQ obtained from a PQQ and glutamic acid [Japanese Patent Publication (Laid-Open) No. 190882/1991]; 2-carbamoylethyl-OPQ obtained from a PQQ and glutamine [Japanese Patent Publication (Laid-Open) No. 188082/1991]; 2-methylthioethyl-OPQ obtained from a PQQ and methionine [Japanese Patent Publication (Laid-Open) No. 19088/1991]; benzyl-OPQ obtained from a PQQ and phenylalanine [Japanese Patent Publication (Laid-Open) No. 190881/1991]; 4-hydroxyphenylmethyl-OPQ obtained from a PQQ and tyrosine [Japanese Patent publication (Laid-Open) No. 9387/1992]; carboxymethyl-OPQ obtained from a PQQ and aspartic acid; carbamoylmethyl-OPQ obtained from a PQQ and asparagine; 4-imidazolylmethyl-OPQ obtained from a PQQ and histidine; 4-aminobutyl-OPQ obtained from a PQQ and lysine; 3-guanidinopropyl-OPQ obtained from a PQQ and arginine; and mercaptomethyl-OPQ obtained from a PQQ and cysteine. As shown above, R group of each of the OPQs basically corresponds to the R group of α-amino acid [R--CH(NH₂)COOH] which is used for substrate to produce one of OPQs. When tyrosine is used as the α-amino acid, OPQ (R═H) is the main product when the pH value of the reaction mixture is low and 4-hydroxyphenylmethyl OPQ (R═CH₂ C₄ H₄ OH) is main product when the value is high.

Salts of these OPQs include alkali metal salts, alkaline earth metal salts, ammonium salts and substituted ammonium salts, which are also effective as the NGF production accelerating agents. Typical examples are the salts of sodium, potassium, magnesium, calcium, ammonium, trimethylammonium, triethylammonium, and triethanolammonium.

Esters of these OPOs include those in which R¹, R² and R³ in the formula mentioned above represent a hydrogen atom, or an alkyl, alkenyl, or benzyl group, which may be same or different, to form mono-, di- or triesters. These OPQs esters can be prepared by a conventional process wherein an OPQS or its salt is allowed to react with an alcohol. The alkyl group may be a methyl or ethyl group, and the alkenyl group may be an allyl group.

These esters of OPQS can also be obtained by a conventional process wherein a PQQ or its salt is allowed to react with an alcohol to give the corresponding PQQ ester, and then the ester is allowed to react with an amino acid or methylamine.

PQQs have been found to function as a coenzyme for methanol dehydrogenase in methanol-metabolizing bacteria. The term, PQQs, used herein means 4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ) and its salts. The PQQs and their esters are represented by the following formula: ##STR2## wherein in R¹, R² and R³ represent a hydrogen or an alkyl, alkenyl, benzyl, propargyl or alkoxycarbonylalkyl group, which may be same or different.

PQQs and their esters actively accelerate NGF production, but they can not increase the NGF level in cerebral cortex, though they exhibit the production accelerating activity to sciatic nerve in animal experiments. Therefore, they are suitably used as therapeutic agents to prevent degeneration of the peripheral nervous system, such as peripheral nervous trauma and diabetic neuropathy.

PQQs employed in this invention can be prepared by any of a number of known organic chemical syntheses [for example, that mentioned in J.A.C.S. Vol. 103, pages 5599-5600 (1981)] and fermentation methods [for example, that mentioned in Japanese Patent Publication (Laid-Open) No. 218597/1989]. PQQs referred to in this invention means PQQ and its salts, such as sodium and potassium salts of PQQ.

In the above formula for PQQ esters, R¹, R² and R³ represent a hydrogen atom, or alkyl, alkenyl, benzyl, propargyl or alkoxycarbomethyl group, which may be same or different, to form mono-, di- or triesters. The alkyl group may be a methyl or ethyl group, and the alkenyl group may be an allyl group.

PQQ triesters are readily synthesized by reaction of a PQQ with an alcohol [see, for example, Japanese Patent Publications (Laid-Open) Nos. 123781/1991 and 145492/1991]. PQQ monoesters or diesters can be obtained by partial hydrolysis of a PQQ triester under basic condition. PQQ diesters can also be obtained by reaction of a PQQ monoester with an alcohol under suitably selected reaction conditions of temperature and time.

OPQs and PQQs and their esters in this invention may be administered orally or non-orally. In case of oral administration, they may be administered in the form of conventional formulations such as capsules, tablets, and powders. In case of non-oral administration, the formulations may be those for injections and parenteral fluids. Sustained release formulations are also effective.

Dosage and dosing time vary depending on symptoms, ages, body weights, dosing formulations, and others, but they may be administered ordinarily in an amount of 1-500 mg a day for adults in the case of oral administration, or 0.1-100 mg in one or several dosage units per day in the case of non-oral administration.

In preparing formulations of the active ingredients of this invention, any additives, such as surface active agents, excipients, coloring agents, preservatives, coating auxiliaries, and the like may be suitably used. They may also be used in combination with other pharmaceuticals.

The following non-limiting examples illustrate the NGF production accelerating activity of PQQs, OPQs and their esters, according to the present invention.

EXAMPLE 1

L-M cells of fibroblast cell line originated from a mouse connective tissue were suspended in a 199 culturing medium (manufactured by Flow Laboratories) containing 0.5% peptone (manufactured by Difco Laboratories), and the suspension was placed in a microplate with 96 flat bottom holes to make the cell numbers of 2×10⁴ /hole, which was then incubated in a CO₂ incubator (at 37° C., in an atmosphere of 5% CO₂ and 95% air) for 3 days. Each incubated liquid was replaced by a 199 medium containing OPQ of each given concentration and 0.5% bovine serum albumin (manufactured by Amour Pharmaceutical) or the same medium containing no OPQ, and incubated in a CO₂ incubator.

After 24 hour incubation, the amount of NGF contained in the supernatant fluid was estimated by enzyme immunoassay [see Korsching and Thoenen, Proc. Natl. Acad. Sci., U.S.A., 80, 3513-3516, (1983)]. The results are shown in Table 1.

                  TABLE 1                                                          ______________________________________                                         Amount of    Amount of NGF                                                                               Relative                                             OPQ added    produced     activity                                             (μg/ml)   (pg/ml)      (%)                                                  ______________________________________                                         0            170          100                                                  3.1          203          119                                                  6.3          278          164                                                  12.5         371          218                                                  25           367          216                                                  50           422          248                                                  100          288          169                                                  ______________________________________                                    

NGF assay

A solution of anti-mouse β-NGF antibody (made by using β-NGF prepared from mouse submaxillary gland as antigen) was dispensed to each hole on a 96 hole microplate made of polystyrene (MS-3496F, manufactured by Sumitomo Bakelite Co. Ltd.) in an amount of 50 μl/hole, and stood for 4 hours at 37° C. The antibody not adsorbed to each hole of the microplate was removed, and each hole was rinsed 3 times with a cleansing liquor. A solution of standard β-NGF (manufactured by Toyobo Co., Ltd.) or a sample solution was dispensed to each hole in an amount of 40 μl/hole, and stood for 18 hours at 4° C. Then, the standard β-NGF or sample solution (incubated supernatant as mentioned above) was removed, and each hole was rinsed 3 times. A solution of anti-β-NGF monoclonal antibody labeled with β-glactosidase (manufactured by Boehringer Mannheim) (40 mU/ml, pH 7.6) was dispensed to each hole in an amount of 50 μl/hole, and stood for 4 hours at 37° C. Then, the enzyme-labeled antibody was removed, and each hole was rinsed 3 times. A solution of 4-methylumbelliferyl-β-D-galactoside (manufactured by Sigma) was dispensed to each hole in an amount of 100 μg/hole, and allowed to react for 1.5 hours at room temperature, and then a 0.2 M glycine-sodium hydroxide buffer (pH 10.3) was dispensed to each hole in an amount of 100 μl/hole to stop the enzyme reaction. Fluorescent intensity of 4-methylumbelliferone produced was estimated using a plate reader, and NGF amount was calculated from the standard curve. The results are shown in Table 1. NGF production accelerating activity of the tested compound was shown as a relative value (%) of the NGF amount produced by cells treated with the testing compound against the NGF amount produced by untreated cells without testing compound.

EXAMPLE 2

Procedure of Example 1 was repeated using hydroxymethyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 2.

                  TABLE 2                                                          ______________________________________                                         Amount of hydroxy-                                                                           Amount of NGF                                                                               Relative                                            methyl-OPQ added                                                                             produced     activity                                            (μg/ml)    (pg/ml)      (%)                                                 ______________________________________                                         0             384          100                                                 0.8           525          137                                                 1.6           554          144                                                 3.1           565          147                                                 6.3           582          152                                                 12.5          593          154                                                 25            670          174                                                 50            708          184                                                 100           786          205                                                 200           982          258                                                 400           1,453        378                                                 ______________________________________                                    

EXAMPLE 3

Procedure of Example 1 was repeated using methyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 3.

                  TABLE 3                                                          ______________________________________                                         Amount of methyl-                                                                            Amount of NGF                                                                               Relative                                            OPQ added     produced     activity                                            (μg/ml)    (pg/ml)      (%)                                                 ______________________________________                                         0             384          100                                                 0.8           525          137                                                 1.6           604          157                                                 3.1           599          156                                                 6.3           665          173                                                 12.5          665          173                                                 25            632          165                                                 50            676          176                                                 100           681          177                                                 200           873          227                                                 400           1,075        280                                                 ______________________________________                                    

EXAMPLE 4

Procedure of Example 1 was repeated using 2-carboxyethyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 4.

                  TABLE 4                                                          ______________________________________                                         Amount of 2-carboxy-                                                                          Amount of NGF                                                                               Relative                                           ethyl-OPQ added                                                                               produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              264          100                                                1.6            330          125                                                3.1            387          147                                                6.3            330          125                                                12.5           349          132                                                25             356          135                                                50             368          139                                                100            381          144                                                200            381          144                                                400            454          172                                                ______________________________________                                    

EXAMPLE 5

Procedure of Example 1 was repeated using benzyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 5.

                  TABLE 5                                                          ______________________________________                                         Amount of benzyl-                                                                              Amount of NGF                                                                               Relative                                          OPQ added       produced     activity                                          (μg/ml)      (pg/ml)      (%)                                               ______________________________________                                         0               264          100                                               1.6             375          142                                               3.1             368          139                                               6.3             375          142                                               12.5            349          132                                               25              375          142                                               50              356          135                                               100             317          120                                               200             298          113                                               400             271          103                                               ______________________________________                                    

EXAMPLE 6

Procedure of Example 1 was repeated using 1-methylpropyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 6.

                  TABLE 6                                                          ______________________________________                                         Amount of 1-methyl-                                                                           Amount of NGF                                                                               Relative                                           propyl-OPQ added                                                                              produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              264          100                                                1.6            442          167                                                3.1            446          177                                                6.3            430          163                                                12.5           418          158                                                25             484          183                                                50             430          163                                                100            381          144                                                200            337          128                                                400            245           93                                                ______________________________________                                    

EXAMPLE 7

Procedure of Example 1 was repeated using 2-methylpropyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 7.

                  TABLE 7                                                          ______________________________________                                         Amount of 2-methyl-                                                                           Amount of NGF                                                                               Relative                                           propyl-OPQ added                                                                              produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              264          100                                                1.6            460          174                                                3.1            400          152                                                6.3            460          174                                                12.5           424          161                                                25             436          165                                                50             478          181                                                100            418          158                                                200            393          149                                                400            324          123                                                ______________________________________                                    

EXAMPLE 8

Procedure of Example 1 was repeated using 2-methylthioethyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 8.

                  TABLE 8                                                          ______________________________________                                         Amount of 2-methyl-                                                                           Amount of NGF                                                                               Relative                                           thioethyl-OPQ added                                                                           produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              264          100                                                1.6            490          186                                                3.1            442          167                                                6.3            484          183                                                12.5           430          163                                                25             466          177                                                50             480          186                                                100            424          161                                                200            356          135                                                400            349          132                                                ______________________________________                                    

EXAMPLE 9

Procedure of Example 1 was repeated using 2-carbamoylethyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 9.

                  TABLE 9                                                          ______________________________________                                         Amount of 2-carbamoyl-                                                                          Amount of NGF                                                                               Relative                                         ethyl-OPQ added  produced     activity                                         (μg/ml)       (pg/ml)      (%)                                              ______________________________________                                         0                382          100                                              0.8              443          116                                              1.6              512          134                                              3.1              505          132                                              6.3              546          143                                              12.5             632          165                                              25               639          167                                              50               652          171                                              100              566          148                                              200              512          134                                              400              408          107                                              ______________________________________                                    

EXAMPLE 10

Procedure of Example 1 was repeated using 1-methylethyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 10.

                  TABLE 10                                                         ______________________________________                                         Amount of 1-methyl-                                                                           Amount of NGF                                                                               Relative                                           ethyl-OPQ added                                                                               produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              382          100                                                0.8            386          101                                                1.6            478          125                                                3.1            492          129                                                6.3            526          138                                                12.5           546          143                                                25             553          145                                                50             606          159                                                100            573          150                                                200            539          141                                                400            485          127                                                ______________________________________                                    

EXAMPLE 11

Procedure of Example 1 was repeated using 4-hydroxyphenylmethyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 11.

                  TABLE 11                                                         ______________________________________                                         Amount of 4-hydroxy-                                                                          Amount of NGF                                                                               Relative                                           phenylmethyl-OPQ                                                                              produced     activity                                           added (μg/ml)                                                                              (pg/ml)      (%)                                                ______________________________________                                         0              382          100                                                0.8            560          147                                                1.6            580          152                                                3.1            553          145                                                6.3            632          165                                                12.5           613          160                                                25             613          160                                                50             652          171                                                100            429          112                                                200            335           88                                                ______________________________________                                    

EXAMPLE 12

Procedure of Example 1 was repeated using OPQ methyl ester at 2-position (OPQ-2-ME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 12.

                  TABLE 12                                                         ______________________________________                                         Amount of      Amount of NGF                                                                               Relative                                           OPQ-2-ME added produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              259          100                                                0.8            290          112                                                1.6            299          115                                                3.1            299          115                                                6.3            344          133                                                12.5           308          119                                                25             234           90                                                ______________________________________                                    

EXAMPLE 13

Procedure of Example 1 was repeated using OPQ methyl ester at 7-position (OPQ-7-ME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 13.

                  TABLE 13                                                         ______________________________________                                         Amount of      Amount of NGF                                                                               Relative                                           OPQ-7-ME added produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              219          100                                                0.8            217           99                                                1.6            265          121                                                3.1            242          111                                                6.3            234          107                                                12.5           235          107                                                25             266          121                                                ______________________________________                                    

EXAMPLE 14

Procedure of Example 1 was repeated using OPQ dimethyl ester at 2- and 7-positions (OPQ-2,7-DME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 14.

                  TABLE 14                                                         ______________________________________                                         Amount of     Amount of NGF                                                                               Relative                                            OPQ-2,7-DME   produced     activity                                            added (μg/ml)                                                                             (pg/ml)      (%)                                                 ______________________________________                                         0             259          100                                                 0.8           299          115                                                 1.6           345          133                                                 3.1           336          130                                                 6.3           317          122                                                 12.5          326          126                                                 25            274          106                                                 ______________________________________                                    

EXAMPLE 15

Procedure of Example 1 was repeated using OPQ dimethyl ester at 2- and 9-positions (OPQ-2,9-DME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 15.

                  TABLE 15                                                         ______________________________________                                         Amount of     Amount of NGF                                                                               Relative                                            OPQ-2,9-DME   produced     activity                                            added (μg/ml)                                                                             (pg/ml)      (%)                                                 ______________________________________                                         0             259          100                                                 0.8           249           96                                                 1.6           290          112                                                 3.1           342          132                                                 6.3           373          144                                                 12.5          435          168                                                 25            393          152                                                 50            258          100                                                 ______________________________________                                    

EXAMPLE 16

Procedure of Example 1 was repeated using OPQ trimethyl ester (OPQ-TME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 16.

                  TABLE 16                                                         ______________________________________                                         Amount of      Amount of NGF                                                                               Relative                                           OPQ-TME added  produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              336          100                                                0.8            508          151                                                1.6            677          201                                                3.1            636          189                                                6.3            628          187                                                12.5           653          194                                                25             563          168                                                50             524          156                                                100            516          154                                                200            587          175                                                400            508          151                                                ______________________________________                                    

EXAMPLE 17

Procedure of Example 1 was repeated using OPQ 2-methyl-7,9-diethyl ester (OPQ-2-ME-7,9-DEE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 17.

                  TABLE 17                                                         ______________________________________                                         Amount of       Amount of NGF                                                                               Relative                                          OPQ-2-ME-7,9-DEE                                                                               produced     activity                                          added (μg/ml)                                                                               (pg/ml)      (%)                                               ______________________________________                                         0               381          100                                               0.8             476          125                                               1.6             558          146                                               3.1             542          142                                               6.3             576          151                                               12.5            716          188                                               25              625          164                                               50              608          160                                               100             421          110                                               ______________________________________                                    

EXAMPLE 18

Procedure of Example 1 was repeated using PQQ-Na₂, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 18.

                  TABLE 18                                                         ______________________________________                                         Amount of     Amount of NGF                                                                               Relative                                            PQQ.Na.sub.2 added                                                                           produced     activity                                            (μg/ml)    (pg/ml)      (%)                                                 ______________________________________                                         0             204          100                                                 0.8           319          156                                                 1.6           302          148                                                 3.1           398          195                                                 6.3           677          332                                                 12.5          1,076        527                                                 25            2,515        1,233                                               50            5,915        2,900                                               100           8,034        3,938                                               200           5,428        2,661                                               400           2,487        1,219                                               ______________________________________                                    

EXAMPLE 19

Procedure of Example 1 was repeated using PQQ dipotassium salt (PQQ-K2), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 19.

                  TABLE 19                                                         ______________________________________                                         Amount of     Amount of NGF                                                                               Relative                                            PQQ.K.sub.2 added                                                                            produced     activity                                            (μg/ml)    (pg/ml)      (%)                                                 ______________________________________                                         0             384          100                                                 0.8           560          146                                                 1.6           554          144                                                 3.1           729          190                                                 6.3           812          211                                                 12.5          1,453        378                                                 25            3,288        856                                                 50            7,047        1,835                                               100           7,281        1,896                                               200           4,425        1,152                                               400           1,237        322                                                 ______________________________________                                    

EXAMPLE 20

Procedure of Example 1 was repeated using PQQ methyl ester at 2-position (PQQ-2-ME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 20.

                  TABLE 20                                                         ______________________________________                                         Amount of      Amount of NGF                                                                               Relative                                           PQQ-2-ME added produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              727          100                                                0.8            1,434        197                                                1.6            1,663        229                                                3.1            2,501        344                                                6.3            3,126        430                                                12.5           4,510        620                                                25             5,611        772                                                50             5,188        714                                                100            2,397        330                                                ______________________________________                                    

EXAMPLE 21

Procedure of Example 1 was repeated using PQQ methyl ester at 7-position (PQQ-7-ME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 21.

                  TABLE 21                                                         ______________________________________                                         Amount of      Amount of NGF                                                                               Relative                                           PQQ-7-ME added produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              219          100                                                1.6            258          116                                                3.1            282          129                                                6.3            493          225                                                12.5           1,238        565                                                25             1,448        661                                                50             928          424                                                100            335          153                                                200            266          121                                                ______________________________________                                    

EXAMPLE 22

Procedure of Example 1 was repeated using PQQ dimethyl ester at 2- and 9-positions (PQQ-2,9-DME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 22.

                  TABLE 22                                                         ______________________________________                                         Amount of     Amount of NGF                                                                               Relative                                            PQQ-2,9-DME   produced     activity                                            added (μg/ml)                                                                             (pg/ml)      (%)                                                 ______________________________________                                         0             727          100                                                 0.8           1,165        160                                                 1.6           1,777        244                                                 3.1           3,126        430                                                 6.3           3,790        521                                                 12.5          4,790        659                                                 25            6,829        939                                                 50            5,499        756                                                 100           4,584        631                                                 200           1,852        255                                                 ______________________________________                                    

EXAMPLE 23

Procedure of Example 1 was repeated using PQQ trimethyl ester (PQQ-TME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 23.

                  TABLE 23                                                         ______________________________________                                         Amount of      Amount of NGF                                                                               Relative                                           PQQ-TME added  produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              341          100                                                0.8            429          126                                                1.6            464          136                                                3.1            600          176                                                6.3            954          280                                                12.5           1,448        425                                                25             2,852        836                                                50             3,015        884                                                100            1,678        492                                                ______________________________________                                    

EXAMPLE 24

Procedure of Example 1 was repeated using PQQ triethyl ester (PQQ-TEE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 24.

                  TABLE 24                                                         ______________________________________                                         Amount of      Amount of NGF                                                                               Relative                                           PQQ-TEE added  produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              381          100                                                0.8            2,190        575                                                1.6            2,160        567                                                3.1            1,889        496                                                6.3            1,882        494                                                12.5           1,249        328                                                25             550          144                                                ______________________________________                                    

EXAMPLE 25

Procedure of Example 1 was repeated using PQQ triallyl ester (PQQ-TAE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 25.

                  TABLE 25                                                         ______________________________________                                         Amount of      Amount of NGF                                                                               Relative                                           PQQ-TAE added  produced     activity                                           (μg/ml)     (pg/ml)      (%)                                                ______________________________________                                         0              204          100                                                0.8            403          198                                                1.6            414          203                                                3.1            611          300                                                6.3            776          380                                                12.5           1,063        521                                                25             707          347                                                ______________________________________                                    

EXAMPLE 26

Procedure of Example 1 was repeated using PQQ triethoxycarbonylmethyl ester (PQQ-TECE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 26.

                  TABLE 26                                                         ______________________________________                                         Amount of       Amount of NGF                                                                               Relative                                          PQQ-TECE added  produced     activity                                          (μg/ml)      (pg/ml)      (%)                                               ______________________________________                                         0               381          100                                               3.1             428          112                                               6.3             455          119                                               12.5            558          146                                               25              669          176                                               50              1,908        508                                               100             3,092        812                                               200             304           80                                               ______________________________________                                    

EXAMPLE 27

Procedure of Example 1 was repeated using PQQ tripropargyl ester (PQQ-TPGE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 27.

                  TABLE 27                                                         ______________________________________                                         Amount of       Amount of NGF                                                                               Relative                                          PQQ-TPGE added  produced     activity                                          (μg/ml)      (pg/ml)      (%)                                               ______________________________________                                         0               381          100                                               1.6             389          102                                               3.1             599          157                                               6.3             764          201                                               12.5            2,453        644                                               25              6,022        1,581                                             50              3,926        1,030                                             100             505          133                                               ______________________________________                                    

EXAMPLE 28

L-M cells were incubated in similar way as in Example 1 using epinephrine which has been known as an NGF production accelerator, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 28.

                  TABLE 28                                                         ______________________________________                                         Amount of     Amount of NGF                                                                               Relative                                            epinephrine   produced     activity                                            added (μg/ml)                                                                             (pg/ml)      (%)                                                 ______________________________________                                         0             179          100                                                 0.8           180          101                                                 1.6           168           94                                                 3.1           180          101                                                 6.3           187          104                                                 12.5          224          125                                                 25            319          178                                                 50            431          241                                                 100           872          487                                                 200           259          144                                                 ______________________________________                                    

Epinephrine showed NGF production accelerating activity at an amount of not less than 12.5 μg/ml, and exhibited the maximum value (about 500%) at an amount of 100 μg/ml. On the other hand, OPQs and OPQ esters showed almost the same degree of NGF production accelerating activity as shown in Examples 1-17. Also, PQQs and PQQ esters gave high activity values even at a lower concentration, having markedly higher NGF production accelerating activity, when compared with epinephrine.

EXAMPLE 29

L-M cells were incubated in similar way as in Example 1.

In three groups (A, B and C) of L-M cell incubation systems, Group A contained no tested compound, Group B contained 100 μg/ml of PQQ.sup.. Na₂, and Group C contained 275 μg/ml of epinephrine. Amounts of NGF produced during the course of times (3, 6, 9, 12, 24, 30, 36 and 48 hours from the beginning of incubation) were estimated. The results are shown in Table 29.

As obvious from the table, addition of PQQ.sup.. Na₂ definitely increases NGF production, as compared with the cases of no addition and adding epinephrine.

                  TABLE 29                                                         ______________________________________                                         Incubating                                                                              Amount of NGF produced (pg/ml)                                        period             (B) PQQ.Na.sub.2                                                                           (C) Epinephrine                                 of time  (A) No    100 μg/ml                                                                               275 μg/ml                                    (hr)     addition  added       added                                           ______________________________________                                          0        63          81        66                                              3       124        180         92                                              6       219        667        202                                              9       254       2,657       288                                             12       271       4,947       347                                             24       378       9,924       556                                             30       420       8,635       689                                             36       458       8,334       873                                             48       465       7,769       1,960                                           ______________________________________                                    

EXAMPLE 30

SD female rats (7 weeks age, 160-190 g) were put under anesthesia by intramuscularly administering 25 mg of ketamine hydrochloride and 0.25 mg of doloperidol. Left femor sciatic nerve was exposed and cut off, and the cut ends were connected with a gap of about 2 mm using a silicone tube (1 mm inner diameter, 6 mm length). The gap between the cut ends was filled with an isotonic sodium chloride solution. After reduction of the operational cut, each 0.5 ml of an aqueous 2% gum arabic solution containing a given concentration of OPQ was administered intraperitoneally. As control, 0.5 ml of an aqueous 2% gum arabic solution containing no OPQ was administered intraperitoneally. As for positive control, the gap of the cut ends of sciatic nerve was filled with an isotonic sodium chloride solution containing 1 mg/ml of NGF, and 0.5 ml of an aqueous 2% gum arabic solution containing no OPQ was administered intraperitoneally. After 4 weeks from the operation, animals were sacrificed by cervical bertebral luxation, and the reproduced sciatic nerves were collected. Cross sectional slices of the reproduced sciatic nerves were prepared and dyed with hematoxylineosine, and number of the reproduced nerve fibers was counted.

The results are shown in Table 30. As obvious from the table, number of the reproduced sciatic nerves was much increased by administering OPQ, which was comparable to direct injection of NGF.

                  TABLE 30                                                         ______________________________________                                         Amount of OPQ   Number of  Relative                                            administered    reproduced activity                                            (μg/kg rat)  sciatic nerves                                                                            (%)                                                 ______________________________________                                          0              12,590     100                                                  5              12,600     100                                                  10             12,848     102                                                  50             13,912     111                                                 100             19,839     158                                                 500             17,870     142                                                 Positive control                                                                               20,932     166                                                 (NGF gap injection)                                                            ______________________________________                                    

EXAMPLE 31

Procedure of Example 30 was repeated using OPQ trimethyl ester (OPQ-TME), in place of OPQ, to estimate the reproduction accelerating activity of OPQ-TME for sciatic nerve. The results are shown in Table 31.

As obvious from the table, number of the reproduced sciatic nerves was much increased by administering OPQ.sup.. TME, which was comparable to NGF.

                  TABLE 31                                                         ______________________________________                                         Amount of OPQ.TME                                                                              Number of    Relative                                          administered    reproduced   activity                                          (μg/kg rat)  sciatic nerves                                                                              (%)                                               ______________________________________                                          0              12,590       100                                                5              11,860        94                                                10             11,193        89                                                50             12,780       102                                               100             22,966       182                                               500             26,313       209                                               Positive control                                                                               20,932       166                                               (NGF gap injection)                                                            ______________________________________                                    

EXAMPLE 32

Procedure of Example 30 was repeated using PQQ.sup.. Na₂, in place of OPQ, to estimate the reproduction accelerating activity for sciatic nerve. The results are shown in Table 32.

As obvious from the table, number of the reproduced sciatic nerves was much increased by administering OPQ.sup.. Na₂, which was comparable to NGF.

                  TABLE 32                                                         ______________________________________                                         Amount of PQQ.Na.sub.2                                                                         Number of    Relative                                          administered    reproduced   activity                                          (μg/kg rat)  sciatic nerves                                                                              (%)                                               ______________________________________                                          0              12,590       100                                                5              16,984       135                                                10             21,822       173                                                50             26,142       208                                               100             18,247       145                                               500             14,031       111                                               Positive control                                                                               20,932       166                                               (NGF gap injection)                                                            ______________________________________                                    

EXAMPLE 33

Procedure of Example 30 was repeated using PQQ trimethyl ester (PQQ-TME), in place of OPQ, to estimate its reproduction accelerating activity for sciatic nerve. The results are shown in Table 33.

As obvious from the table, number of the reproduced sciatic nerves was much increased by administering PQQ-TME, which was comparable to NGF.

                  TABLE 33                                                         ______________________________________                                         Amount of PQQ-TME                                                                              Number of    Relative                                          administered    reproduced   activity                                          (μg/kg rat)  sciatic nerves                                                                              (%)                                               ______________________________________                                          0              12,590       100                                                5              19,047       151                                                10             21,054       167                                                50             24,940       198                                               100             17,882       142                                               500             17,902       142                                               Positive control                                                                               20,932       166                                               (NGF gap injection)                                                            ______________________________________                                    

EXAMPLE 34

OPQ trimethyl ester (OPQ-TME) was suspended in 0.5 ml of an aqueous 2% gum arabica in a given concentration, and the suspension was administered to Wistar male rats (8-10 weeks age, 200-250 g) intraperitoneally. Administrations were conducted every other days once a day, 4 times in total. After 2 days from the last administration, rats were dissected under anesthesia, and the neocortex, submaxillary grand and hippocampus were collected. The following procedures were conducted under ice-cooling. Each of these tissues was weighed, and mixed with a 20 time-volume of a phosphate buffer (8 g/l of NaCl, 0.2 g/l of KCl, 1.15 g/l of Na₂ HPO₄ and 0.2 g/l of KH₂ PO₄), and the mixture was homogenized by an ultrasonic crusher, followed by centrifugation at 10,000×G for 30 minutes to separate the supernatant. Amount of NGF contained in the supernatant was estimated by enzyme immunoassay. The results are shown in Table 34. NGF amount (ng) per 1 mg (wet weight) of neocortex, submaxillary gland or hippocampus was set forth as the average value ± standard error from 3 heads tested simultaneously. As for former two tissues, relative activities were also shown against the case of no OPQ-TME administration.

As obvious from the table, NGF contents in neocortex and submaxillary gland were increased by administering OPT-TME. Particularly, the degree of increase was high in neocortex.

                                      TABLE 34                                     __________________________________________________________________________            Neocortex   Submaxillary gland                                                                         Hippocampus                                     Amount of                                                                             NGF         NGF         NGF                                             OPQ-TME                                                                               (ng/mg Relative                                                                            (ng/mg Relative                                                                            (ng/mg                                          administered                                                                          tissue activity                                                                            tissue activity                                                                            tissue                                          (μg/kg rat)                                                                        wet weight)                                                                           (%)  wet weight)                                                                           (%)  wet weight)                                     __________________________________________________________________________     0      1.83 ± 0.05                                                                        100  0.63 ± 0.04                                                                        100  2.44 ± 0.23                                  0.1    2.64 ± 0.17                                                                        144  0.70 ± 0.05                                                                        111  2.31 ± 0.20                                  0.5    2.60 ± 0.40                                                                        142  0.85 ± 0.06                                                                        135  2.20 ± 0.46                                  1.0    3.17 ± 0.44                                                                        173  0.67 ± 0.09                                                                        106  --                                              __________________________________________________________________________

EXAMPLE 35

Procedure of Example 34 was repeated using PQQ trimethyl ester (PQQ-TME), in place of OPQ-TME, to estimate the accelerating activities of PQQ-TME for NGF contents of neocortex, submaxillary gland and hippocampus. The results are shown in Table 35.

Administration of PQQ-TME did not increase the NGF contents in neocortex, submaxillary gland and hippocanpus. Supposedly, PQQ-TME have no NGF production accelerating activity to central nervous system.

                  TABLE 35                                                         ______________________________________                                         Amount of                                                                               NGF production amount                                                 PQQ-TME  (ng/mg tissue wet weight)                                             administered         Submaxillary                                              (mg/kg rat)                                                                             Neocortex   gland       Hippocampus                                   ______________________________________                                         0        2.51 ± 0.23                                                                             1.42 ± 0.07                                                                             2.53 ± 0.23                                0.1      2.25 ± 0.10                                                                             1.67 ± 0.15                                                                             2.44 ± 0.04                                0.5      2.19 ± 0.33                                                                             1.58 ± 0.07                                                                             2.10 ± 0.15                                1.0      2.71 ± 0.30                                                                             1.30 ± 0.08                                                                             --                                            ______________________________________                                    

Thus, as OPQs and their esters exhibit NGF production accelerating activity, and, in animal experiments, they increase the NGF content in neocortex, and accelerate reproduction of sciatic nerve, the nerve growth factor production accelerators of the present invention are suitably utilized as preventive and therapeutic agents for functional disorders of central nervous system, particularly, Alzheimer's dementia, cerebral ischemia and spinal trauma, as well as for functional disorders of peripheral nervous system, particularly, peripheral nervous system trauma and diabetic neuropathy.

Further, as PQQs and their esters exhibit strong NGF production accelerating activity, and, in animal experiments, they accelerate the reproduction of sciatic nerve, the present accelerators are suitably utilized as preventing and treating agents for functional disorders of peripheral nervous system, particularly, peripheral nervous system trauma, diabetic neuropathy, etc. 

What we claim is:
 1. A method for accelerating the production of nerve growth factor in a non-diabetic host in need of such acceleration using a compound which is oxazopyrroloquinoline or an ester thereof represented by the formula: ##STR3## wherein R represents a hydrogen atom or an alkyl group having 1-4 carbon atoms, which may be substituted with a hydroxyl, carboxyl, mercapto, carbamoyl, hydroxyphenyl, guanidyl, imidazolyl, or methylmercapto group, and R¹, R² and R³ represent a hydrogen atom or an alkyl, alkenyl or benzyl group, which may be the same or different, which method comprises administration to such host of an amount of such compound which is effective for such acceleration.
 2. The method of claim 1, wherein the acceleration of the production of nerve growth factor is effective for the treatment of degeneration of the central or peripheral nervous system in a non-diabetic host in need of such treatment.
 3. A method as in claim 2 wherein the compound is oxazopyrroloquinoline.
 4. A method as in claim 2 wherein the compound is hydroxymethyl-oxazopyrroloquinoline.
 5. A method as in claim 2 wherein the compound is methyloxazopyrroloquinoline.
 6. A method as in claim 2 wherein the compound is 1-methylpropyl-oxazopyrroloquinoline.
 7. A method as in claim 2 wherein the compound is 2-methylthioalkyl-oxazopyrroloquinoline. 